Determination of Chloral Hydrate, Trichloroacetic Acid, and Trichloroethanol PAUL J. FRIEDMAN and JACK
R. COOPER
Department o f Pharmacology, Yale University School o f Medicine, New Haven, Conn.
,Studies on the enzymatic metabolism of chloral hydrate necessitated the development of simple, specific, and sensitive assays of the drug and its metabolites. The procedures outlined are based on some hitherto unreported absorption bands produced when chloral hydrate, trichloroacetic acid, and trichloroethanol are heated with alkali in the presence of pyridine. The use of these new absorption bands results in a more sensitive determination of the compounds and a direct determination of trichloroethanol without resorting to other procedures. The methods reported provide further insight into an understanding of the chemistry of this reaction.
S
to characterize the enzymes involved in the oxidation and reduction of chloral hydrate necessitated the development of suitable analytical procedures for the estimation of the drug and the products of its metabolic alteration: trichloroacetic acid and trichloroethanol. Traditionally, chloral hydrate and trichloroacetic acid have been determined by modifications of the Fujiwara reaction, all of which involve heating the compound with pyridine in the presence of strong base, and lead to the production of a crimson color with an absorption maximum around 540 mp. The literature on the Fujiwara reaction has been reviewed comprehensively by Set0 and Schultze (6). Several methods have been used for the determination of trichloroethanol, which, according to previous investigators, does not give a colored product under the conditions of the Fujiwara reaction (1, 6). Butler ( 1 ) separated the compound by multiple solvent extractions, followed by oxidation with dichromate t o trichloroacetic acid, which then was determined by the Fujiwara reaction. Marshall and Owens (3) observed that trichloroethanol heated in alkaline solution is decomposed to formaldehyde. They separated the trichloroethanol by djstillntion, decomposed it, and determined the formaldehdye. Set0 and Schultze (6) oxidized trichloroethanol with chromic oxide by incubation a t TUDIES
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ANALYTICAL CHEMISTRY
37" C. for 24 hours in concentrated nitric acid, and used the Fujiwara reaction in the determination of the trichloroacetic acid formed. Glazko, Dill, Wolf, and Kazenko ( 2 ) also determined chloral hydrate and trichloroacetic acid by measuring the crimson color produced in the Fujiwara reaction using a low temperature and long heating time. Trichloroethanol mas determined essentially by the procedure of Marshall and Owens ( 3 ) . The method described is based on the observation that trichloroacetic acid, chloral hydrate, and trichloroethanol all react with pyridine upon heating in alkali to form a chromophore with an absorption maximum a t 370 mp. This band is three to four times as intense as that a t 540 mp produced by chloral hydrate and trichloroacetic acid and is much more stable. Trichloroethanol also produces a visible color in this reaction, a yellow band with an absorption maximum a t 440 mH. Because this band is not given by the other compounds, it is used to determine trichloroethanol directly. Trichloroacetic acid is determined a t 370 mp by heating a sample with alkali before adding pyridine, a procedure which ensures the destruction of trichloroethanol and chloral hydrate, but does not seriously reduce the absorption resulting from trichloroacetic acid. Chloral hydrate is then determined by difference, subtracting that part of the absorbance a t 370 mp due to trichloroethanol and trichloroacetic acid from the total.
certified reagent grade pyridine is satisfactory when fresh, but repeated exposure to air will cause the material to yield a decreased extinction coefficient for trichloroethanol a t 440 mp with rapid fading of the color. No effect on the 370-mp absorption band is observed. The depressed absorption a t 440 mp may be restored to normal by adding a small amount of either chloral hydrate or trichloroacetic acid (as little as 0.02 pmole) to the trichloroethanol solution to be determined. This observation has been used as the basis for a simple check on the purity of the pyridine. In practice, the procedure of apportioning a fresh bottle of Fisher pyridine into several small bottles has been used. As only one small bottle is opened a t a time, the stock is protected. The potassium hydroxide solution used is 10 molal. Preparation of Tissues. The methods outlined are applicable to the determination of the trihalogenated compounds in all types of tissue preparations, but with crude undialyzed, liver homogenates the absorption of the chromophore is reduced about 25%. As standards are run with every determination, this defect is corrected automatically. Sulfosalicylic acid, 5% final concentration, was used to precipitate proteins prior to the determination. Tissue blanks ranged from zero to 0.01 pmole of trichloroacetic acid per 44 mg. of tissue depending on the type of preparation, such as acetone powder extract or dialyzed supernate.
REAGENTS
A. I n a test tube of borosilicate glass, 5.0 ml. of pyridine are added to 1.0 ml. of a solution containing 0.02 to 0.2 pmole of chloral hydrate or trichloroacetic acid and 0.1 to 0.8 pmole of trichloroethanol. The contents of the tube are mixed, 2.0 ml. of 10 molal potassium hydroxide are added, and the solutions are mixed again. The tube is placed in a boiling water bath for exactly 4 minutes and transferred immediately to an ice bath for 3 to 5 minutes, Then 3 ml. of the pyridine layer are pipetted into a test tube containing 0.5 ml. of water. The contents of the tube are transferred t o a cuvette and read immediately in a Beckman spectrophotometer a t 370 and 440 mp. When a number of sam-
Trichloroacetic acid and chloral hydrate were obtained commercially and assayed by titration according t o U.S. Pharmacopeia XV, with the difference that 0.1 rather than 1.ON sodium hydroxide and hydrochloric acid were used. Trichloroethanol was a gift from E. R. Squibb and Sons, obtained through the courtesy of W. A. Lott. A small amount of trichloroethanol was also prepared by reduction of chloral hydrate with sodium borohydride. The product was collected by distillation under reduced pressure. The purity of the pyridine used in the determinations is very important. Either redistilled pyridine or Fisher
PROCEDURE
2 00-
,001 1 I
W A V E L E N G T H , MP
Figure 1 . Absorption spectra of chloral hydrate and trichloroacetic acid 0
340
Figure 2.
ples are to be determined, the solutions in the test tubes are kept in a n ice bath until they are assayed. B. For trichloroacetic acid determination, another 1.0-ml. aliquot of the soiution is mixed with 2 ml. of 10 molal potassium hydroxide in a test tube and placed in a boiling water bath for exactly 2 minutes. The tube is placed immediately in a n ice bath for 2 to 3 minutes, and then 5.0 ml. of pyridine are added and mixed with the solution in the test tube. Thp tube is placed in a boiling water bath for exactly 4 minutes. The remainder of the procedure follows as in Procedure A except that only the absorption a t 370 mp is determined. These solutions are read against a check cell of 3.0 ml. of pyridine and 0.5 ml. of n-ater. Standards are run simultaneously with unknowns, I n Procedure A, 0.1 pmole of chloral hydrate or trichloroacetic acid gives an average absorbance of 0.52 +37& With preheating in Procedure B, trichloroacetic acid absorption is reduced 10 to 15y0. Trichloroethanol, 0.1 pmole, gives a n average of 0.12 5 5 % absorbance units a t 440 mp and 0.17 =t3y0a t 370 mp. I n a mixture of the three compounds, the error in the determination of trichloroacetic acid and trichloroethanol will be about +3y0 n-hile with chloral hydrate the error averages +5%. Calculations. The concentrations of t h e three compounds in t h e unknown mixture may be calculated as follom: Let (TCA)
=
TCA370.4
=
TCAam
=
concentration of trichloroacetic acid absorbance of TCA sample in Procedure A a t 370 m
p
absorbance of TCA sample in Procedure B a t 370 mp
TCA* = Btandard TC-4 solution TCA" = unknown TCA solution and similarly for trichloroethanol (TCE) and chloral hydrate (CH)
and because
= tota1"sio~TCE2370* - TC-4'37o~
CH2370*
by substitution, (CH)"
=
CH'HOA (CH)s CH'aio~
EXPERIMENTAL
Absorption Spectra. Figures 1 and 2 show t h e spectra of samples produced by heating each of t h e three compounds with pyridine in alkali. The extinction coefficients of chloral hydrate and trichloroacetic acid are t h e same within experimental error a t 370 mp, while that of trichloroethanol is about one third as great. Proportionality. A linear relationship between absorbance and concentration holds for chloral hydrate a n d trichloroacetic acid over a t least a tenfold range of concentration. With trichloroethanol there is a slight reduction in extinction coefficient with concentrations belov about 0.2 pmole per ml. If a sample a t such a concentration is encountered, it is advisable to run simultaneously a standard which gives a comparable absorbance. Stability of Chromophores. The absorption band of t h e three compounds a t 370 mp decreases no more than 25YGduring a 10-minute period a t room temperature. Under the same conditions, the 440-mp absorption of trichloroethanol decreases by about 6%. Water, rather than acid, alkali, or alcohol, was the best diluent of pyridine
390 440 W A V E LENGTH, MIJ
540
490
Absorption spectrum of trichloroethanol
with respect to stability of the chomophores. The addition of 0.5 nil. of watt'r is sufficient to remove the turbidity of the pyridine layer. On standing, the pyridine will become cloudy. This may be retarded by capping the cuvettes with their glass covers and may be reversed by diluting with water. If in studies of tissues in vitro a n aliquot of more than 0.1 ml. of a crude homogenate was analyzed, the resultant pyridine layer was more cloudy than with simple aqueous solutions. Clarification for reading !vas obtained by adding 1.0 instead of 0.5 ml. of water. Heating Time. Although a 4-minute heating is insufficient t o reach a plateau a t 370 mp, for the three halogenated compounds, it is close to the optimum heating time for trichloroethanol at 440 mp, which is about 3.5 minutes. After this time the 440mp band decreases in intensity. Alkali Concentration. Increasing t h e concentration of t h e alkali reagent above 10 molal will increase t h e sensitivity of t h e test, but the decomposition of trichloroethanol and chloral hydrate is performed best with only moderately stiong alkali. Also, it is desirable t o use a minimum of reagents for simplicity. Preheating with dilute alkali (0.1-11) is adequate to destroy chloral hydrate and trichloroethanol derivatives that give a 370-mp absorption, with minimal effect on trichloroacetic acid absorption, However, trichloroethanol gives no 440mp band with dilute alkali. Strong alkali (16 to 17 molal), n-hich develops a n intense 440-nip band, cannot effectively remove chloral hydrate and trichloroethanol components of the 3TOmp reading without reducing the trichloroacetic acid absorption significantly. A 10 molal potassium hydroxide solution does not affect trichloroacetic acid sensitivity seriously, but it does VOL. 30, NO. 10, OCTOBER 1958
0
1675
remove chloral hydrate and trichloroethanol components with 2 minutes of preheating and is strong enough to produce a good color with trichloroethanol at 440 mp. Different concentrations of alkali can be used for optimal determination of these substances individually. DISCUSSION
Although the mechanism of the Fujiwara reaction is not yet understood: it has been observed by the authors and other investigators (4) to be specific for compounds containing at least three halogen atoms on one carbon atom. It is not clear whether the two absorption maxima given by each compound represent competing reactions, or two peaks of a single complex. However, with trichloroethanol the absorption of the two bands can be varied independently by changing the heating time or alkali concentration. It is always advisable to run standards with a set of unknowns and not to consider the extinction coefficients as constants. As shown in the experimental section,
the conditions of the procedures were chosen as a compromise designed to simplify measuring all three compounds. If only chloral hydrate or trichloroacetic acid were to be determined, a heating time of 6 to 8 minutes would be preferable, because it would give a greater absorption at 370 mp than the 4-minute time prescribed in the method. HOKever, the 440-mp absorption of trichloroethanol decreases in intensity after 3.5 minutes of heating. The absorption of trichloroacetic acid and chloral hydrate is strictly additive; however, that of chloral hydrate and trichloroethanol is not. This error is less marked at low concentrations of chloral hydrate and trichloroethanol; its cause is unknown. I n practice, the estimate of chloral hydrate in a mixed sample will be on the low side. Interest here is in accurate measurement of the metabolites which have been formed in experiments in vitro, and the remaining chloral hydrate concentration is of secondary importance. The amount of chloral hydrate remaining is far greater than that of the metabolites formed,
and this greatly reduces the significance of the error. To obtain more accurately the Concentration of chloral hydrate in a mixture of the three compounds, readings of the crimson color a t 540 mp may be made in Procedure A. Then chloral hydrate concentration is given by the difference after absorption due to trichloroacetic acid is subtracted; this may be calculated from Procedure B. LITERATURE CITED
(1) Butler, T. C., J . Pharmacol. Esptl. Therap. 92,49 (1948). (2) Glazko, A. J., Dill, W. A., Wolf, L. M.,Kaeenko, A,, Zbid., 121, 119 (1 9.57). ,. \ - - -
(3) Marshall, E. K., Jr., Owens, A. H., Jr., Bull. Johns Hopkins Hosp. 95, 1 (19W). (4) Ross, J. H., J . Biol. Chem. 58, 641 ( 1923). ( 5 ) Seto, T. A,, Schultze, Ll. 0.) ASAL. CHEST. 28,1625 (1956).
RECEIVED for review November 22, 1957. Accepted June 18, 1958. Taken from the thesis to be submitted by Paul J. Friedman in partial fulfillment of the requirements for the degree of doctor of medicine, Yale University.
Separation of Glycerol from a Polyhydric Alcohol Mixture by Nonionic Exclusion RA
T. CLARK
Forest Products laboratory, Forest Service, U. S. Department of Agriculture, Madison, Wis.
,Column chromatography with an ion exchange resin was used to separate glycerol from a mixture of polyhydric alcohols that resulted from the hydrogenolysis of glucose or sorbitol. Other fractions obtained included sorbitol and erythritol, each with traces of xylitol, and a mixed fraction of ethylene glycol and propylene glycol. The method may be useful for commercial application.
T
separation of simple mixtures of polyhydric alcohols by selective extraction with acetone or other solvents, or by codistillation with chloroform or hydrocarbons, has been reported ( 5 ) . These separations, however, are slow and incomplete. The separation of glycerol from triethylene glycol and other nonionic materials, by passage of their solution through columns of ion exchange resin, has been described by Wheaton and Baumann (9). The separation has been explained on the basis of the extent and rate a t M-hich individual solute constituents of a
mixture are distributed between the solvent (water) inside the resin particles, and that outside, during passage through the resin column. Distribution constants, K d ,for a number of nonionic materials and resins have been reported. This method was used to investigate the separation of a mixture of polyhydric alcohols resulting from the hydrogenolysis of glucose or sorbitol ( 2 ) . The mixture, after removal of traces of ionic impurities by ion exchange, had the following analysis.
HE
1676
ANALYTICAL CHEMISTRY
70 Sorbitol Xylitol Erythritol Glycerol 1,2-Propanediol Ethylene glycol Water (approximately)
8 87 0 69 2 90 11 60 7 65 4 34 60 00
Traces of ethanol, methanol, mannitol, and other compounds were also present. After glycols were removed by distillation a t reduced pressure, glycerol could not be satisfactorily separated from the mixture by distillation a t a pressure
of 0.5 mm. of mercury, without decomposition of the nondistillable sugar alcohols. The steam distillation and extraction methods cited were unsatisfactory for separating the glycerol from the sugar alcohols. I n the resin column separation, the extent and rate of distribution of a solute between the water phases depend upon such factors as temperature, and the type, ionic form, particle size, and cross linkage of a resin. Other factors determining effectiveness of separations are column-flow rate, column height, and concentration and volume of column feed (9). The effects of several of these variables were studied in a series of experiments to determine the applicability of the method to the large scale separation of a glycerol fraction from the hydrogenolysis mixture, and to permit recycle of sugar alcohols in further hydrogenolysis to glycerol. As reported by Wheaton and Baumann (9) Dowex Type 50 H + resin was used for the experiments on the basis of distribution constants because the best
